Nipah virus (NiV), a highly pathogenic member of the Paramyxoviridae family, was first isolated and identified after a 1998-1999 outbreak of fatal encephalitis among pig farmers and abattoir workers in Southeast Asia. NiV and its close relative, Hendra virus, have been classified as Biosafety Level 4 (BSL-4) select agents due to their broad host range, their numerous routes of transmission, and the high rates of mortality associated with infection. Despite recent advances in understanding the cellular tropism of NiV, there is currently no prophylaxis available for animals or humans, and treatment remains primarily supportive. Several DNA and virus-like particle (VLP)-based vaccines are under development and have been demonstrated to induce high-titer (103) antibody responses that protect various animal models against live NiV challenge. As an alternative approach, we are employing antibodies against NiV glycoprotein (NiV-G) to affinity-select peptide mimotopes from a complex random sequence library displayed on VLPs of MS2 bacteriophage.
Virus-like particles (VLPs) are derived from self-assembly of the structural proteins of a virus. Vaccines can be made from VLPs in two different ways. First, a vaccine against a given virus can be made from the structural proteins of that virus. In such cases, the VLP serves as a non-infectious version of the virus itself, and is utilized to safely elicit an antibody response to the virus from which it is derived. The existing vaccines for hepatitis B virus and human papilloma virus are examples. Second, VLPs can serve as scaffolds for the immunogenic presentation of epitopes derived from other sources (from whatever source, viral or not). The VLP-based vaccines described in this application are in this second category. There are made by the immunogenic presentation of Nipah Virus-derived epitopes on the surface of a VLP made by self-assembly of the coat protein of bacteriphage MS2 expressed from a plasmid in E. coli. In this case the VLP merely serves as a carrier to present a NiV epitope (or epitope mimic) to the immune system in an immunogenic format.
Because VLPs are structurally repetitious, linking target antigens, either genetically or chemically, to the VLP surface causes them to be displayed at high densities. Such densely-spaced repetitive elements provoke efficient oligomerization of the membrane-associated immunoglobulin (Ig) molecules that constitute the B cell receptor (BCR), which in turn promotes increased stimulation of the B cell. Thus, a normally non-immunogenic substance (e.g. a peptide) becomes strongly immunogenic when displayed as a dense repetitive array on a VLP. MS2 VLPs self-assemble from 180 copies of a single coat protein into a 28-nm icosahedron. The vaccines described here use a VLP assembled from a single-chain coat protein dimer, and can display up to 90 peptides per particle in a highly accessible surface loop. Thus presented, peptide epitopes, are able to elicit high titer antibody responses in immunized animals or humans, even though they are very poorly immunogenic on their own. MS2 VLPs are, furthermore, highly tolerant of random peptide insertions and encapsidate the mRNA that encodes the coat protein-random peptide fusion, enabling their development as a display platform for a wide range of different peptide sequences, and enabling the construction of random peptide libraries from which specific epitope mimics can be isolated by affinity-selection on antibody targets. In addition to its dependence on the ability to tolerate a wide variety of different peptide insertions, affinity-selection also takes advantage of the fact that the VLP encapsidates the mRNA that encodes it and any guest peptide it carries, thus allowing the recovery of affinity-selected sequences by reverse transcription and PCR amplification.
We have employed complex (˜1010 members), random peptide (10 amino acids in length) libraries to affinity-select MS2 VLPs that bind to neutralizing monoclonal antibodies against NiV-G. After two rounds of selection at high valency (90 peptides/VLP) and two rounds at low valency (˜3 peptides/VLP), we obtained peptides for each antibody that map directly onto the NiV-G sequence. VLPs that display a high density (90 copies/VLP) of these peptides bind to their respective antibodies with high affinity (Kd=1-20 nM). Immunization of mice with the resulting VLPs elicits antibodies that neutralize infection by a Nipah G-protein pseudotyped vesicular stomatitis virus (a surrogate for Nipah Virus itself).